As a better understanding of cellular processes at the molecular level is gained, novel materials can be designed at the nanometer length scale which exploits this new knowledge to effectively deliver therapeutic compounds. However, limitations in synthesizing such small entities often preclude the ability to rationally incorporate multiple functions, which in turn restricts available delivery strategies. Significantly, our proposed delivery system can uniquely address some of these limitations of small nanoscale systems, thereby potentially extending the feasibility of nanoscale delivery systems. The goal of this proposed investigation is to incorporate multiple functionalities into 25-nm protein nanoparticles and test their potential for targeted drug delivery in cancer cells. This can potentially increase the effectiveness of a given drug by significantly decreasing the amount of drug needed, expanding its therapeutic capabilities, allowing greater control over targeting and release, and decreasing side effects. Our synthesis strategy involves genetically designing chimeras of protein nanoparticles, which enables the nanoscale architecture to be specifically tailored in a relatively straightforward manner. This proposed work will, for the first time, investigate the response of breast cancer cells to these protein nanoparticles and test the feasibility of using these multifunctional scaffolds for improving therapeutic delivery of doxorubicin. We hypothesize that cytotoxicity in cancer cells will be greatest when the multiple properties of cellular targeting, drug encapsulation, and pH-responsive dissociation and drug release are combined within each protein nanoparticle. To test this hypothesis, we propose the following specific aims: (1) Fabricate drug-loaded, pH-responsive protein nanoparticles that display cancer-targeting ligand, (2) Determine optimal conditions for cytotoxicity and in vitro dose-response profiles in breast cancer cells, and (3) Reconcile the mechanisms of endocytosis, compartmental trafficking, and drug release with nanoparticle characteristics and cytotoxicity. This proposed work not only will develop the technology of multifunctional protein nanoparticles in drug delivery, but will enable the use of the E2 scaffold as a model for determining general delivery principles. This includes identifying the structures and properties of targeted nanoparticles which affect cellular interaction and behavior. Furthermore, our target disease in this proposed work is breast cancer, which will be treated with protein-encapsulated doxorubicin. Although doxorubicin is a conventional line of treatment, it exhibits dose-dependent cardiotoxicity in patients. Information learned from this investigation could identify general strategies in nanoparticulate drug delivery that increase cytotoxicity in only cancer cells while decreasing overall doses and severe side-effects. PUBLIC HEALTH RELEVANCE: Multifunctional Protein Nanocapsules for Targeted Delivery In creating a new class of nanoscale drug carriers, the scope of novel therapeutic strategies in therapeutic delivery will be broadened. This has the potential to expand the efficacy of disease treatment and promote regenerative medicine.